The enzyme pyruvate decarboxylase will be studied as representative of the class of enzymes requiring thiamine pyrophosphate and Mg(II). The two principal objectives are the following: 1. Mechanistic aspects: search for the rate limiting step on the enzyme (12C/13C kinetic isotope effects on CH3CHO release); definition of the role of the amino group of the coenzyme in catalysis (Transition State analogs and high resolution nmr); definition of the catalytic and binding amino acid residues (employing fluorescent coenzyme analogs, chemical modification, Eu(III)-fluorescence) and to see if the four subunits are all catalytic or not. 2. Conformational aspects to establish the conformation of enzyme bound coenzyme and the distance between the enzyme bound coenzyme and Mn(II) ion (as a replacement for the required Mg(II) ion by high resolution and relaxation magnetic resonance studies).